1. Field of the Invention
The present invention relates generally to the fields of lipid biochemistry and liposomes. More particularly, the invention provides amphiphilic molecules that incorporate a hydrophilic material or polymer attached to two or more spatially distinct hydrophobic residues. On contact with water, these amphiphilic molecules display surface activity and self-assemble into multimolecular aggregates and liquid crystalline phases. The invention thus also provides liposomes of enhanced stability that incorporate such amphiphilic molecules, and methods of using these formulations in a variety of applications in the fields of drug delivery, nutrition, bio-diagnostics, cosmetics, blood products and related applications.
2. Description of Related Art
Amphiphilic molecules are so named because the structures contain hydrophilic and lipophilic (hydrophobic) parts. The molecules distribute across air-water and oil-water interfacial boundaries and display surface activity. In oil and water mixtures, these help form and stabilize emulsions and co-dissolve other materials. When dispersed in water at concentrations above critical solubility limits, these can be induced to self assemble into a variety of spatially ordered molecular aggregates including micelles and lamellar bilayers which can entrap other molecules in the lipid and/or the aqueous compartments of the aggregates. Amphiphile-containing emulsions, micelle, and lipid lamellar bilayer aggregates are important vehicles for parenteral delivery of therapeutic agents and nutrients.
Liposomes are spherical vesicles of self-closed hydrated bilayers of amphiphilic lipids surrounding a generally central inner aqueous phase core which can differ in composition from the extraliposomal aqueous medium (Bangham and Horne, 1964). The lipid chains may be liquid-crystalline or solid-like gel phases. Liposomes are colloidal particles ranging in diameter from 20 nm to 5000 nm. Depending on the size and the number of constituent lamellar layers, these are classified as small or large unilamellar vesicles, and as multilamellar vesicles. The multilamellar vesicles have additional water layers trapped adjacent to the hydrophilic ends (polar head groups) between the regular dual arrays of the lipophilic (hydrophobic) alkyl chains (fatty tails).
The lipid bilayer of the unilamellar vesicles is akin in composition and structure to the outer membrane of eukaryotic cells. The vesicle bilayer provides a significant controllable barrier to the movement of various molecules and ions between the inner aqueous core and the bulk aqueous phase surrounding the liposome (Bangham et al., 1965). This barrier function is paramount in many applications including drug delivery vehicles.
The need for and importance of a functional barrier is well illustrated by potential applications of liposomes in enzyme replacement therapies for inherited metabolic diseases, in other therapies using bioactive peptides and proteins, and in hemoglobin-based blood substitutes. The safety and efficacy of therapeutic/bioactive proteins depends upon their ability to overcome metabolic and transport barriers and reach the target site in a biologically active form. This in turn is dependent on the route for administration.
In general, exogenous proteins cause immunogenic and antigenic reactions and undergo rapid hydrolytic degradation in vivo. These problems can be partially alleviated by modification of the protein by covalent conjugation to a biocompatible hydrophilic polymer such as a monofunctional polyethyleneglycol (PEG). As an example, adenosine deaminase conjugated to xcfx89-methyl-polyethyleneglycol (MePEG) of average molecular weight 5000 shows lower immunogenicity and antigenicity, and prolonged blood circulation half-life. However, although the conjugate has seen some clinical use, it has relatively low enzyme activity (Beauchamp et al., 1983). Despite the loss of bioactivity on covalent modification of proteins that is known to occur generally, other MePEG-linked proteins, including the inimunoregulatory cytokine interleukin-2, and the oxygen transporter hemoglobin, are being investigated for ultimate use in vivo.
The extra effort needed to develop reasonably active conjugates for every enzyme contemplated for in vivo is a significant limitation in this field. This could be avoided if an encapsulation process applicable to all unmodified enzymes could be developed, but this has yet to be achieved. Potential systemic and transdermal delivery systems for unmodified bioactive proteins, such as entrapment in biodegradable microspheres fabricated from poly(lactide-co-glycolide) and other polymers, are being investigated (Gombotz and Pettit, 1995). Liposomal systems have also been proposed and, although these offer the advantage that they are capable of self-assembly (Gregoriadis, 1988), they currently suffer from certain drawbacks.
Liposomes are normally prepared from natural phospholipids and synthetic analogues such as the electrical charge neutral zwitterionic phosphatidylcholines. Minor proportions of anionic phospholipids, such as phosphatidylglycerols, are added to generate a net negative surface charge for colloid stabilization. The lipid chains in the bilayer may be present as crystalline or mesophase (liquid-crystalline) states. The type of mesophase controls the physical integrity of the liposomes in vitro and in vivo, and liposomes with gel phase bilayers are more stable in blood than those with liquid-crystalline bilayers.
For liposomes administered parenterally, the blood circulation half-life, distribution and disposition in organs and tissues is correlated strongly with the diameter and the surface properties of the liposomes. Most liposomes are rapidly taken up by the phagocytic cells of the reticuloendothelial system (RES), the circulating mononuclear phagocytic cells and those located in the liver and spleen, and their blood circulation half-lives are short (a few minutes). This uptake is generally mediated by binding of plasma proteins (opsins) to the liposomal surface. The non-specific xe2x80x9cscavenger receptorxe2x80x9d that recognizes negative charges in large arrays may be involved also (Nishikawa et al., 1990). Liposomes smaller in diameter than the average diameter of the fenestrae in the blood capillaries leak out. The average diameter of the fenestrae in the imperfectly formed sinusoids in rapidly growing tumors is larger than in normal tissues and therefore liposomes smaller than about 100 nm in diameter migrate into tumors.
The above two proclivities provide the basis for targeting liposome-encapsulated drugs to liver and tumors respectively. A primary requirement for targeting therapies for metabolic disorders to other organs and tissues is that liposomes be able to evade uptake as above and have long blood circulation half-life. The inclusion of ganglioside GM1, a natural glycolipid with terminal sialic acid residue, as a minor envelope component improves circulation life, presumably because of changes in the liposomal surface characteristic (Allen and Chonn, 1987). However, this has yet to yield sufficiently beneficial results.
Recently, certain xcfx89-methyl-polyethyleneglycol-conjugated anionic lipids have been developed, notably xcfx89-Me-PEG-phosphatidylethanolamines (MePEG-PE), and used as envelope components at about 5 mole % of total lipid. The resulting liposomes display pendant MePEG residues on the outer lipid envelope surface, and these are considered to act as steric barriers to opsin attachment and RES uptake (Lasic et al., 1991; Needham et al., 1992; Woodle and Lasic, 1992). Therefore, these liposomes are called sterically stabilized liposomes. The degree of polymerization and surface density of the MePEG, and anionic charge are important parameters for liposome stability. However, even with optimum parameters, these sterically stabilized liposomes only have a blood circulation half-life of between about 12 and about 48 hours, as compared to the blood circulation half-life of red blood cells of 28 days.
Antibodies and receptor-specific ligands have also been tethered to the surface of sterically stabilized liposomes for inducing targeted delivery to specific tissues. Counterproductively, the pendant MePEG chains in these mixed surface ligand type liposomes not only prevent uptake by the RES but also hinder the approach and binding of liposome-surface-linked antibodies to the target tissue receptors. Therefore, the art is, at best, ambivalent about their potential for targeted delivery to specific tissues. For instance, according to an authoritative comment xe2x80x9cthe remaining problems of accessibility of a particular tissue and cells as well as overlooked severity of triggering an immune response to the host organism by antibody or lectin-coated liposomes makes this goal rather remote at presentxe2x80x9d (Lasic and Barenholz, 1996).
In an attempt to generate liposomes of improved stability, two broad trends are discernible in the current research literature. There is continuing interest in (i) optimizing existing liposome types, and (ii) in developing alternative systems. Optimization is being attempted by the development of alternatives lipid conjugates of MePEG. The preparation and applications of diacylglycerol and cholesterol in place of PE, and for the MePEG-PE series, the investigation of different spacers and chemistries for conjugating MePEG to PE have been described (Parr et al., 1994; Zalipsky, 1995). However, these have still not countered the prevailing pessimism explained above. Alternatively, various hydrophilic polymers other than MePEG have been conjugated to PE, but only poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline) conjugates have afforded even the minimum protection from hepatosplenic uptake seen with MePEG-PE (Woodle et al., 1994).
Data on the retention and chemical stability of MePEG-lipid conjugates incorporated into unilamellar vesicles are available. Many MePEG-lipid stabilized liposomes gave very little improvement in circulation half-life, and this was traced to the rapid removal of the hydrophilic coating. The latter is attributed to the loss of the intact MePEG-lipid from the liposomal membrane. Significant chemical breakdown of MePEG-lipid (MePEG-PE) occurs, especially after its detachment from the liposome body. Finally, the loss of hydrophilic coating precedes liposome clearance (Parr et al., 1994).
Focusing on the properties inherent in the MePEG-lipid conjugate structure, the present inventor has reevaluated these data in terms of relative propensity of the conjugate for retention in the liposome bilayer and proclivity for migration into the extraliposomal fluid, and, the mole % of MePEG-lipid in the bilayer phospholipids needed for complete coverage with an adequate depth of MePEG chains. For representative phospholipid structures, conjugates with relatively short MePEG chains tend to stay in the bilayer but provide inadequate surface barrier. Conjugates with longer chains tend to migrate into the surrounding aqueous medium, particularly at high concentrations which provide adequate surface barrier. The inventor realized that the balance of these opposing influences/conflicting constraints cannot be improved further without a radical departure from the structures typified by MePEG-lipid. In particular, these efforts cannot provide the minimum one order of magnitude increase in half- life needed for encapsulated functional proteins such as hemoglobin for blood substitutes.
The present invention overcomes the foregoing and other drawbacks inherent in the prior art by providing a range of new synthetic amphiphilic materials of a unique molecular structural class. The structural motif of the amphiphilic materials of the invention results from the combination of a hydrophilic residue, compound or polymer, that is attached to two or more hydrophobic residues or moieties at spatially distinct sites on the hydrophilic residue. In certain embodiments, the two or more hydrophobic moieties are attached to the hydrophilic residue at distinct sites of an intermediate or wide spatial distance, such that they are attached at xe2x80x98spatially distantxe2x80x99 sites.
The hydrophilic compound or polymer is preferably non-immunogenic, for example, a polyethylene glycol, and may additionally contain functional groups for attaching agents other than the hydrophobic residues. The hydrophobic residues or moieties are preferably lipids, such as phosphatidyl, that can intercalate in the lipid bilayer array of conventional liposomes. The hydrophobic moieties are preferably covalently attached to the hydrophilic compound or polymer, such as by using an ester, ether, or other selected bond-type.
The amphiphilic materials of the invention form lyotropic self-assembled multimolecular aggregate phases alone and in mixtures with conventional amphiphiles (for instance phosphatidylcholines) on interaction with aqueous media. The hydrated hydrophilic polymer segments of the molecular structure are oriented around the lipid segment of the lyotropic self-assembly in a topography reminiscent of canopies and staples.
The novel amphiphiles provided by the invention and the derived self-assembled aggregates have utility generally as processing-aids, e.g., emulsifiers, and as functional ingredients in products fabricated as lipid- microemulsion-, micelle-, and liposome-based protective delivery vehicles for use in agriculture, diagnostics, drug, food, nutrition, personal-care and hygiene products, and, industrial applications.
The unique interfacial topography makes these novel amphiphilic materials and the derived self-assembled aggregates particularly appropriate for application in liposomal and micellar preparations suitable for passive and targeted drug delivery and antigen-presentation for diagnostics. The unique topography may be engendered additionally by equilibrating the hydrated novel amphiphiles with preformed liposomes and biological cells to create non-immunogenic red blood cells for blood substitutes and analogous biomaterials.
Accordingly, the present invention provides an amphiphilic molecule comprising a hydrophilic compound, polymer or polymer residue having attached, at spatially distinct sites, at least two hydrophobic moieties. The hydrophilic compounds of these molecules may be polymers or polymeric residues, such that the compounds are constructed from repeating units of individual monomers; may be co-polymers comprised of ordered or random repeating units of two or more individual monomers; or may be any other suitable hydrophilic compound that permits the operative attachment of the hydrophobic moieties.
The two or more hydrophobic moieties are attached to the hydrophilic compound at xe2x80x9cspatially distinct sitesxe2x80x9d. The spatially distinct attachment means that the at least two hydrophobic moieties must be attached to distinct attachment sites of the hydrophilic compound or polymer, and are not formed by extending a first hydrophobic moiety by direct attachment to a second hydrophobic moiety (forming a single extended chain). The hydrophobic moieties or residues are therefore appended to the hydrophilic compound or polymer, and are not attached to each other.
The spatially distinct attachment sites on the hydrophilic compound are sites that are adapted to receive the hydrophobic moieties. This means that the attachment sites are capable of physical and functional attachment to a hydrophobic moiety and are separated from one another such that the attachment of a first hydrophobic moiety to a first attachment site on the hydrophilic compound does not impair the physical and functional attachment of a second hydrophobic moiety to the nearest or second attachment site on the hydrophilic compound.
In certain embodiments, it may be that the spatially distinct attachment sites are separated by only a single carbon atom. In other embodiments, the hydrophobic moieties or residues will be attached to the hydrophilic compound or polymer at spatially distinct sites of increasing distance, which distances may be termed as short, moderate, intermediate, long or maximal distances. In general, short spatial distances will be on the order of between about 1, 2, 3, 4 or 5 carbon atoms in length, or the equivalent distance as spanned by other chemical constituents of the hydrophilic compound or polymer. The spatially distinct attachment sites may be separated by lengths directly corresponding to or equivalent to lengths of carbon atoms of between about 5, 10, 15, 20, 25, 50, 100, 150, 250, 500, 1000 or so.
The spatially distinct attachment sites may also be described in reference to their geometrical location along a hydrophilic compound or polymer. For example, the attachment sites may be located at one or more terminal portions of a hydrophilic compound or polymer, generally proximal to such terminal compounds, spaced equidistant along a hydrophilic compound or polymer, clustered in one region of the hydrophilic compound or polymer, randomly distributed along the hydrophilic compound or polymer, and the like. The spatially distinct attachment sites may also be understood with reference to each of FIG. 1, FIG. 3, FIG. 4, FIG. 7 and FIG. 8.
In certain preferred embodiments, the two or more hydrophobic moieties will be attached at spatially distinct sites in order to impart a defined property to the resultant amphiphilic molecule. In other embodiments, the hydrophobic moieties may be randomly attached throughout a hydrophilic compound in order to prepare an amphiphilic molecule with a plurality of hydrophobic moieties. In the first group of embodiments, one may elect to attach two or more hydrophobic moieties to attachment sites that are separated by moderate or intermediate distances, such that the resultant amphiphilic molecules will be able to associate with a lipid bilayer in a desired manner. For example, attachments at, proximal to, or in the region of the termini of a hydrophilic compound or polymer will result in an amphiphilic molecule capable of integrating into a bilayer generally as shown in FIG. 5.
Those of skill in the art will appreciate that, in general, attaching two or more hydrophobic moieties to a defined spatial region of a hydrophilic compound will promote closer association of that portion of the hydrophilic compound to a lipid bilayer upon admixture. Equally, widely separating the hydrophobic moieties at spatially distant attachment sites will result in an amphiphilic molecule in which the hydrophilic compound is more likely to be more flexibly associated with a bilayer or to have intervening regions that are able to migrate to some distance of the bilayer (either in loops or as inverted tear-drops), while the amphiphilic molecule as a whole remains in association with a bilayer by virtue of attachment through said two or more hydrophobic moieties.
In certain embodiments, the invention provides an amphiphilic molecule comprising a hydrophilic compound, polymer or polymeric residue and at least a first and a second hydrophobic moiety or residue, the first and second hydrophobic moieties being attached to said hydrophilic compound at distinct first and second attachment sites. As set forth above, the first and second attachment sites may be proximal to each other, at intermediate distances along the polymer, or widely distant from each other, including being distant from each other for the entire length of the molecule such that they are attached at each terminus.
The amphiphilic molecules of the invention or xe2x80x9camphiphilesxe2x80x9d will preferably include a biocompatible hydrophilic compound or polymeric residue when the resultant amphiphile is intended for use in veterinary, medicinal or other biomedical applications. By the term xe2x80x9cbiocompatiblexe2x80x9d, is meant that the hydrophilic compound and the resultant amphiphilic molecule do not elicit significant adverse or untoward reactions upon administration to the particular animal in which they are intended for use. Determinations of biocompatibility are readily made by those of ordinary skill in the art, and include assessing the known properties of compounds, as described in the scientific literature, prior to generating an amphiphilic molecule and the testing of the molecule in appropriate in vitro and in vivo studies.
It will be appreciated that different biocompatible polymers and resultant amphiphiles may be prepared for use in distinct embodiments. For example, for use in cosmetic or other external medical or veterinary applications, the polymer and amphiphile are required to be generally dermatologically safe and not to elicit significant adverse reactions upon contact or repeated contact with the skin. Similar concerns are applied to amphiphiles for internal use, during which use they should not cause significant systemic or local toxicity in the animal or human subject. Such considerations apply equally to the hydrophobic moieties for attachment to create the resultant amphiphile.
The hydrophilic compounds, polymers or polymer residues for use in the invention may be substantially linear, linear, partially branched, highly branched or a pendant or star hydrophilic compound or polymer. Suitable examples of such polymers are described herein, with particular reference to the appended figures and to Table 1. Any of the individual compounds listed within Table 1 and known to those of skill in the art may be further joined to create a hybrid or conjugate hydrophilic compound.
To be xe2x80x9chydrophilicxe2x80x9d in the context of the present invention, the compound or polymer is required to have measurable solubility in water, preferably of at least about 10%, 20%, 30% or so, or even greater, and should also preferably be soluble in certain organic solvents.
The hydrophilic compound or polymer components of the amphiphiles will preferably have an average molecular weight of between about 100 and about 100,000 daltons, with all intermediate molecular weights between these ranges being contemplated. For example, an appropriate hydrophilic compound for use herewith may have an average molecular weight of about 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 75,000 and about 100,000 or more. In certain preferred embodiments, the molecular weight of the hydrophilic component will be between about 100 and about 20,000, between about 100 and about 10,000, between about 2,000 and about 20,000, between about 2,000 and about 10,000, or between about 100 and about 10,000 or so.
In certain preferred embodiments, the hydrophilic compound of the amphiphile will be a polyethylene glycol (PEG). Other preferred compounds are polyvinyl pyrrolidone and polyethyleneimine.
The xe2x80x9chydrophobic moieties or residuesxe2x80x9d are preferably hydrophobic components that are sterically compatible with typical lamellar lipid bilayers, such that they are capable of spontaneously forming bilayers, or intercalating into bilayers, and are generally selected so as to achieve a good steric fit without significant perturbation of the normal packing geometry of lamellar bilayers. A wide variety of such molecules are known to those of skill in the art and are suitable for use herewith.
In general terms, hydrophobic residues of short, medium or long lipid chains may be employed. Short chain lipids generally have less than about eight carbon atoms (8C). Where such short chain hydrophobic moieties are employed, it may be advantageous to employ a moderate amount of such residues in order to provide an amphiphile molecule with a plurality of appended short chain lipids.
In more preferred embodiments, it is believed that advantages will result from the use of hydrophobic residues having between about 8 carbon atoms (8C) and about 26 carbon atoms (26C), with those having between about 12 carbon atoms (12C) and about 20 carbon atoms (20C) being generally preferred.
A wide variety of hydrophobic compounds are available in the art, any one or more of which may be used to advantage in the present invention. These are exemplified by single, double, multiple, linear and branched chain hydrophobic moieties. Certain exemplary hydrophobic moieties are included herewith in Table 2. For example, the hydrophobic moiety may be an alkylether, fattyester, dialkylglycerol, alkylamino, dialkylamino, diacylglycerol, sphingolipid, synthetic cationic lipid precursor, sterol, cholestanic acid, a phospholipid or perfluoro analog thereof. In other embodiments, one or more or a combination of the following compounds may be employed: 1,2-distearoylglycerol or 1,2-dioleoylglycerol, ceramidophosphoric acid or O-acetyl-ceramidophosphoric acid, 1,2-dioleyl-3-dimethylaminopropanediol, 1,2-dimyristoyl-3-dimethylaminopropanediol, cholesterol or xcex2-sitosterol, distearoyl phosphatidic acid, dioleylphosphatidic acid, a bisphosphatidyl glycerol, phosphatidylethanolamine or a phosphatidylinositol.
In the amphiphilic molecules of the invention, the hydrophilic compound may be operatively attached to only two hydrophobic moieties. In other embodiments, the hydrophilic compound may be attached to at least 2, at least 3, at least 5, at least 10, at least 15 or at least about 20 or so hydrophobic moieties. The hydrophilic compound or polymer may thus be attached to 2, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or to about 30 or to about 40 or so hydrophobic moieties. The amphiphiles thus include ranges of hydrophobic appendages of between 2 or about 3 and about 40 or so, 30 or so, 20 or so; between about 5 and about 20, between about 5 and about 10, and all intermediates within the foregoing stated ranges.
In certain embodiments, the invention provides amphiphilic molecules comprising a hydrophilic compound or polymer that is operatively attached to a plurality of hydrophobic moieties. In attaching a plurality of hydrophobic moieties or residues to a given hydrophilic compound, those of ordinary skill in the art will appreciate, in light of the present disclosure, that a balance is to be achieved between the hydrophilic and hydrophobic moieties in order to achieve an amphiphile with the desired properties. A particularly preferred property is the ability of the resultant amphiphile to achieve a desired micellar or lamellar mesophase upon interaction with an aqueous medium, such as water.
The invention includes amphiphilic molecules of a bipodal nature, in which the amphiphile comprises only two hydrophobic moieties. Such bipodal amphiphiles may comprise a substantially linear or linear hydrophilic compound or polymer comprising a first and second hydrophobic moiety, the first and second hydrophobic moieties being separately attached to spatially distant attachment sites on said hydrophilic compound, including those embodiments in which the hydrophobic moieties are separately attached to the first and second terminus of the hydrophilic compound, respectively. In other bipodal embodiments, only one of said at least two hydrophobic moieties need be attached at, proximal to, or substantially at one terminus of the hydrophilic compound. Linear polymers need not be used, but these may be preferred for simplicity.
Tripodal amphiphilic molecules are also provided, in which a hydrophilic compound has a first, second and third hydrophobic moiety separately attached thereto. The tripodal amphiphiles may have a three-way symmetry, in which the amphiphilic molecule comprises a branched hydrophilic compound having at least three termini, and wherein the first, second and third hydrophobic moieties are separately attached at, or proximal to, three of said at least three termini.
Oligopodal amphiphiles are also provided, which comprise at least about five or so hydrophobic moieties in conjunction with said hydrophilic compound. The oligopodal compounds may comprise a linear or substantially linear hydrophilic base, or may comprise a moderately branched hydrophilic compound or polymer. In embodiments where a branched compound is employed, five or ten or so hydrophobic moieties may be attached at the termini resulting from each branching chain.
In a similar manner, the invention provides polypodal amphiphilic molecules comprising a plurality of hydrophobic moieties attached to a hydrophilic compound. The compound may again be linear, although the polypodal embodiments are well served by the use of a branched or star hydrophilic compound (e.g., FIG. 4). Again, where a branched or star hydrophilic polymer is employed, a plurality of termini will be available for attachment to a hydrophobic moiety. Depending on the degree of branching of the hydrophilic compound, at least about 10%, 20%, 50%, 75%, 90% or 95% of said termini may be separately occupied by a hydrophobic molecule. Polypodal amphiphiles are also contemplated in which every terminus of a branched or star hydrophilic compound is occupied by an attached hydrophobic moiety.
It will be appreciated that the amphiphilic molecules may comprise two or more identical hydrophobic moieties up to and including a plurality of hydrophobic moieties each of the same molecular type. Alternatively, the amphiphiles may comprise two non-identical hydrophobic species, again up to and including a plurality of hydrophobic species in which each particular appended hydrophobic molecule is of a separate molecular type. All such variations are encompassed within the scope of the invention.
In preferred embodiments, it is contemplated that at least one of said hydrophobic residues will be covalently attached to said hydrophilic compound or polymer residue. In other preferred embodiments, each of said at least two hydrophobic moieties will be attached to the hydrophilic compound via a covalent bond.
Therefore, the invention provides, in part, an amphiphilic molecule comprising a hydrophilic compound having attached, at spatially distinct or distant sites, at least two hydrophobic moieties, wherein at least one of said two hydrophobic moieties are covalently attached to the hydrophilic compound. Also provided are amphiphiles comprising a hydrophilic compound having covalently attached, at spatially distinct or distant sites, at least two hydrophobic moieties. In embodiments where more than two hydrophobic moieties are included within the amphiphile, any two or more of such residues may be covalently attached, and the invention extends the covalent attachment of each hydrophobic moiety to the hydrophilic supporting compound.
However, covalent attachment is not the only means of attaching the two or more hydrophobic moieties to the core hydrophilic compound. For example, non-covalent attachment means may be employed, such as those depending on ionic salt interactions or molecular adducts, such as provided by the tight binding of biotin and avidin. Other receptor-ligand interactions may also be employed. The covalent attachment of at least one of the hydrophobes is particularly preferred, and the covalent attachment of at least two hydrophobes is also a preferred aspect of the invention.
The covalent attachment may be via a direct covalent bond or via a xe2x80x9cmolecular spacerxe2x80x9d, such as a peptide spacer or other bridging molecule. In certain embodiments, one or more of the hydrophobic moieties may be attached to the central hydrophilic compound via a biologically releasable bond. Such bonds include those metabolizable by non-specific chemical or enzymatic pathways, and also those preferentially cleavable under certain conditions, such as occurs upon localization to a specific biological site within the body.
It will also be appreciated that more stable covalent bonds may be used to advantage in formulations that are intended for oral administration or for industrial applications. For example, the alkyl ether type linkages are relatively stable, particularly to hydrolytic enzymes, and are suitable for use in such embodiments.
The range of preferred covalent bonds contemplated for use herewith include alkylamine, alkylamonium, carbamate, amide, ether, ester and phosphodiester bonds.
In certain embodiments, the hydrophilic compound or polymer residue will be first derivatized to introduce at least one functional group permitting the attachment of at least one of said hydrophobic moieties via a covalent bond. The hydrophilic compound may thus be derivatized to introduce at least one aldehyde, thiol, alkyl, dialkylamino, amino, carboxyl, or polyol functional group. In certain embodiments, the derivatization of a hydrophilic compound to produce a methanesulfonate ester of an alcohol group present on the hydrophilic compound is particularly preferred.
In further embodiments, the hydrophilic compound may further comprise a selected agent attached at a site distinct from said at least two hydrophobic moieties. Exemplary agents that may be attached in this manner are antibodies and antigens against which one desires to raise a humoral or cellular immune response. Other appropriate molecules are ligands for biological receptors, or in reciprocal embodiments, one or more biological receptor molecules.
An advantageous aspect of the present invention is that the amphiphilic molecules disclosed herein spontaneously form liquid crystalline multimolecular aggregates upon hydration. Therefore, when formulated with a population of like molecules in an aqueous solution, the novel amphiphiles of the invention self assemble into molecular aggregates and supra molecular assemblies, including micelles, monolayers, bilayers, multimolecular aggregates, lipid microemulsions formulated into a micelle, monolayer, bilayer, multimolecular aggregate, lipid microemulsion, oil globules, fat globules, wax globules, synthetic microreservoirs and liposomes. The self assembly or formulation into such macromolecular assemblies is a surprising feature of this invention. Therefore, the amphiphiles or populations thereof may be advantageously contacted with or dispersed within solvents or liquids, including polar liquids, such as water-based solutions. They may also be formulated in non-polar solvents or liquids.
In certain preferred embodiments, the invention is defined as providing amphiphilic molecules comprising a hydrophilic compound having covalently attached, at spatially distant sites, at least two hydrophobic moieties, said amphiphilic molecule forming a liquid-crystalline multimolecular aggregate upon contact of a number of said amphiphilic molecules with an aqueous solution, wherein the mesophases of said liquid-crystalline multimolecular aggregates, as characterized by X-ray diffraction, include the fluid Lxcex1 and gel Lxcex2 phases.
In still further embodiments, the invention thus provides liquid-crystalline multimolecular aggregates comprising a plurality of amphiphilic molecules dispersed in an aqueous solution, said amphiphilic molecules each comprising a hydrophilic compound having attached, at spatially distinct sites, at least two hydrophobic moieties. Preferably, the liquid-crystalline multimolecular aggregate is one wherein the mesophases of the liquid-crystalline multimolecular aggregates, as characterized by X-ray diffraction, include the fluid Lxcex1 and gel Lxcex2 phases.
In other embodiments, the amphiphiles of the invention may be advantageously mixed or otherwise combined in non-covalent association with at least one distinct amphiphilic, hydrophilic or hydrophobic molecule or population thereof. The physical association of the claimed amphiphiles with populations of distinct lipid components also leads to the spontaneous assembly into multimolecular aggregates, including liposomes. Accordingly, the invention provides formulations wherein one or more of the amphiphiles of the invention is formulated with at least one or more lipid components to form a liposome or lipid complex with at least one liposome bilayer. These embodiments include formulations of small unilamellar vesicles of between about 30 and about 100 nm in diameter, large unilamellar vesicles of between about 100 and about 1,000 nm in diameter, and intermediates thereof, and also includes formulation into multilamellar vesicles.
In certain preferred embodiments, the invention provides amphiphilic molecules formulated with at least one or more lipid components to form a liposome comprising at least an outer liposome bilayer, wherein the hydrophilic compound of the amphiphilic molecule is in contact with at least a portion of said outer liposome bilayer and wherein the hydrophobic moieties of said amphiphilic molecule extend into the outer liposome bilayer. Further examples are amphiphilic molecules that comprise a plurality of hydrophobic moieties that extend into the outer liposome bilayer, wherein the hydrophilic compound extends in contact over a substantial portion of the outer liposome bilayer.
Again, the resultant liposome may be advantageously combined with other surface-available components, such that the liposome comprises at least one surface-available antibody, antigen or binding ligand dispersed in the liposome bilayer or tethered to a component of the liposome bilayer.
In yet further embodiments, the invention provides liposomes, lipid complexes or populations thereof that comprise an amphiphilic molecule that comprises a hydrophilic compound positioned over at least a portion of the outer surface of said liposome or lipid complex, the hydrophilic compound having attached, at spatially distinct sites, at least two hydrophobic moieties that extend into the hydrophobic bilayer of said liposome or lipid complex. The liposomes or complexes may also comprise amphiphilic molecules that comprise a plurality of hydrophobic moieties that extend into the hydrophobic bilayer of the liposome, wherein the hydrophilic compound is positioned over a substantial portion of the outer surface of said liposome or lipid complex. In certain embodiments, the hydrophile will extend over the complete outer surface.
In light of the ability of the amphiphilic molecules themselves to advantageously form liposomes, it will be appreciated that the liposomes of the invention include those in which the liposomal bilayer components are made up of between about 1% amphiphile and about 99-100% amphiphile. Liposomes are contemplated that comprise any amount of novel amphiphiles between the stated range, such that they may comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 44%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and about 100% amphiphile in their bilayer. In such embodiments, the preferred mode of assessing the percentage contribution of amphiphile is to use a mole percent (mole %). In certain preferred embodiments, the amphiphiles will be present in amounts between about 5 mole % and 80 mole %, between about 10 mole % and about 70 mole %, and between about 20-30-40 mole % and about 60 mole % of the total components in the liposome bilayer.
The liposomes of the invention may be formulated with any one or more of the lipid components known to those of ordinary skill in the art. By way of example only, one may mention phospholipids, such as phosphatidylcholine; sterols, such as cholesterol, sphingolipids, such as sphingomyelin; and other components such as sucrose. By means of an exemplary embodiment only, the amphiphile-containing liposomes of the invention may contain the following constituents: between about 40 mole % and about 60 mole % amphiphile; between about 20 mole % and about 30 mole % of phosphatidylcholine; between about 5 mole % and about 10 mole % of sphingomyelin; with the optional addition of other components such as gangliosides and sucrose. Again, the liposomes may comprise in their outer bilayer one or more surface-available components such as antibodies, antigens, binding ligands, receptors, or functional portions thereof. These components may be dispersed within the bilayer or covalently attached to a component thereof.
Any of the liposomes of the invention may be advantageously combined with one or more selected agents. The use of liposomes to deliver agents in medicinal embodiments is known to those of ordinary skill in the art. Accordingly, the liposomes of the invention, comprising the novel amphiphiles in any appropriate amount, may further comprise a fat soluble, water-insoluble agent or a water-soluble agent. It will be appreciated that the selected agent may be encapsulated, entrapped or otherwise physically and functionally associated with the liposome or a lipid complex or multimolecular lipid aggregate. For example, a selected hydrophilic agent may be dispersed within the aqueous phase or lumen of the liposome. Equally, a selected hydrophobic agent may be encapsulated, entrapped or otherwise dispersed within the lipid phase of the liposome. It will be appreciated that multimolecular aggregates may not comprise a lumen as such, i.e., that liposomes are not always spherical in nature and do not define a central chamber in which a selected agent will always be located.
The type of selected agent that may be functionally associated with the liposomes of the invention is virtually limitless and those of skill in the art are referred to exemplary Tables 3A, 3B and 4. By way of example only, one may mention selected pharmacological agents, such as chemotherapeutic agents or cytotoxins (Table 3B); agents to combat infectious organisms, such as antibiotics, anti-virals and fungicides, particularly amphotericin B; immunological components, such as antibodies or fragments thereof, antigens, cytokines and anti-inflamniatory agents in general; enzymes, hormones and neurotransmitters, anesthetics; blood components such as hemoglobin and coagulants; and a variety of nucleic acid molecules, constructs or vectors, including those that express any of the foregoing components and those that include antisense nucleic acids and ribozymes.
In other embodiments, the selected agents may be a nutritional supplement, such as a parenteral fat emulsion, e.g., for use in critically ill animals or patients. In still further embodiments, the liposomes may comprise contrast agents as selected agents. Agents may also be employed for non-medicinal uses, and, by way of example only, one may mention pheromones.
In addition to liposomes, the present amphiphilic molecules may be used in conjunction with biological cells to form amphiphile-coated cells, such as red blood cells. In such embodiments, the amphiphilic molecule provides an outer barrier to the cell in a similar manner, wherein the hydrophilic component of the amphiphilic molecule is in contact with at least a portion of the outer surface of the cell and wherein the hydrophobic appendages extend into the outer membrane of the cell thereby anchoring the amphiphilic molecule in functional association with the cell. Red blood cells from various animals and human subjects are particularly contemplated for use in such embodiments.
In that the amphiphiles of the invention provide important barrier functions to the liposomes, complexes or cells that they are associated with, the invention also provides liposomes with increased half-life, such that the liposomes exhibit a half-life of between about one day and about five or about ten days upon incubation in a buffered solution or in a serum sample in vitro. Even half-lives of over about 48 hours or so are advantageous in comparison to half-lives obtainable with the compositions of the prior art. Techniques for analyzing the half-life of a liposome in vitro and in vivo are well known to those of ordinary skill in the art and are further disclosed in detail herein.
The present invention also provides a number of methodological embodiments. First provided is a method of making an amphiphilic molecule, comprising attaching, or preferably covalently attaching, at least two hydrophobic moieties to spatially distinct or distant attachment sites of a hydrophilic compound or polymer.
Methods of making liposomes are also provided, which generally comprise admixing, in an excess of an aqueous solution, a population of lipid components with a population of amphiphilic molecules, preferably prehydrated amphiphilic molecules, that comprise a hydrophilic compound having at least two hydrophobic moieties attached at spatially distinct sites, the admixing being effective to form a liposome. In such embodiments, the admixing is generally conducted in an aqueous solution, such as water, comprising an effective amount of each of the lipid components and amphiphilic molecules, for a period of time effective and in a manner conducive to form liposomes. Those of skill in the art will understand that the admixing manner most effective to form liposomes involves the use of sonication. The sonication is performed for a period of time effective and in a manner conducive to form liposomes that are coated by the hydrophilic component of the amphiphile, and in which the amphiphile is secured to the liposome by virtue of the hydrophobic moieties extending into their liposomal bilayer.
The present invention also provides analogous methods of making amphiphilic material-coated liposomes, complexes or biological cells, comprising contacting a liposome or biological cell with an amphiphilic material that comprises a hydrophilic compound having at least two hydrophobic moieties attached at spatially distinct sites, such that the hydrophobic moieties extend into the hydrophobic bilayer of said liposome or cell and the hydrophilic compound is positioned over at least a portion of the surface of the liposome or cell. The preferred biological cells for coating with amphiphiles are red blood cells.
The invention further provides methods for encapsulating or entrapping a selected agent in a liposome, comprising admixing a selected agent with a population of liposomes or lipid complexes that comprise an amphiphilic molecule that comprises a hydrophilic compound positioned over at least a portion of the outer surface of the liposome or complex, the hydrophilic compound having attached, at spatially distinct sites, at least two hydrophobic moieties that extend into the hydrophobic bilayer of the liposome or complex, wherein the admixing procedure is effective to cause encapsulation or entrapment of the selected agent in the liposome or lipid complex.
The foregoing methods of the invention lead to even further compositions, such as kits, cosmetic formulations and medicinal delivery compositions. These are generally provided as a kit comprising, in suitable container means, an amphiphilic molecule comprising a hydrophilic compound having attached, at spatially distinct sites, at least two hydrophobic moieties; or a liposomal formulation or lipid complex comprising said amphiphilic molecule. Such kits may further comprise one or more selected agents.
The cosmetic formulations are those comprising, in a cosmetically acceptable base, a population of liposomes or lipid complexes that comprise an amphiphilic molecule that comprises a hydrophilic compound positioned over at least a portion of the outer surface of the liposome or lipid complex the hydrophilic compound having attached, at spatially distinct sites, at least two hydrophobic moieties that extend into the hydrophobic bilayer of the liposome or lipid complex.
The medicinal delivery compositions comprise, in a pharmaceutically acceptable vehicle, a population of liposomes or lipid complexes comprising a selected agent; wherein said liposomes comprise an amphiphilic molecule that comprises a hydrophilic compound positioned over at least a portion of the outer surface of the liposome or lipid complexes, the hydrophilic compound having attached, at spatially distinct sites, at least two hydrophobic moieties that extend into the hydrophobic bilayer of the liposome or lipid complexes.
In yet still further methodological embodiments, the invention provides methods for administering a selected agent to an animal or human, comprising administering to the animal or human a medicinal delivery composition comprising, in a pharmaceutically acceptable vehicle, a population of liposomes or lipid complexes comprising the selected agent; wherein the liposomes or lipid complexes comprise an amphiphilic molecule that comprises a hydrophilic compound positioned over at least a portion of the outer surface of the liposome or lipid complexes, the hydrophilic compound having attached, at spatially distinct sites, at least two hydrophobic moieties that extend into the hydrophobic bilayer of the liposome or lipid complex. These delivery methods may be used to supply any of the selected agents described herein and equivalents thereof, including nutritional supplements, blood components, immunological components, anticancer treatment agents, anti-infectious formulations, anesthetics, enzymes, hormones or neurotransmitters for replacement therapy, and nucleic acid molecules encoding any of the foregoing components.